Membrane-anchored substrate binding proteins are deployed in secondary TAXI transporters.

Substrate-binding proteins (SBPs) are part of solute transport systems and serve to increase substrate affinity and uptake rates. In contrast to primary transport systems, the mechanism of SBP-dependent secondary transport is not well understood. Functional studies have thus far focused on Na+-coupled Tripartite ATP-independent periplasmic (TRAP) transporters for sialic acid. Herein, we report the in vitro functional characterization of TAXIPm-PQM from the human pathogen Proteus mirabilis. TAXIPm-PQM belongs to a TRAP-subfamily using a different type of SBP, designated TRAP-associated extracytoplasmic immunogenic (TAXI) protein. TAXIPm-PQM catalyzes proton-dependent α-ketoglutarate symport and its SBP is an essential component of the transport mechanism. Importantly, TAXIPm-PQM represents the first functionally characterized SBP-dependent secondary transporter that does not rely on a soluble SBP, but uses a membrane-anchored SBP instead.

Expression, purification and preliminary characterisation of the choline transporter LicB from opportunistic bacterial pathogens.

Many opportunistic bacteria that infect the upper respiratory tract decorate their cell surface with phosphorylcholine to support colonisation and outgrowth. These surface modifications require the active import of choline from the host environment, a process thought to be mediated by a family of dedicated integral membrane proteins that act as choline permeases. Here, we present the expression and purification of the archetype of these choline transporters, LicB from Haemophilus influenzae. We show that LicB can be recombinantly produced in Escherichia coli and purified to homogeneity in a stable, folded state using the detergent n-dodecyl-ß-d-maltopyranoside. Equilibrium binding studies with the fluorescent ligand dansylcholine suggest that LicB is selective towards choline, with reduced affinity for acetylcholine and no apparent activity towards other small molecules including glycine, carnitine and betaine. We also identify a conserved sequence motif within the LicB family and show that mutations within this motif compromise protein structure and function. Our results are consistent with previous observations that LicB is a specific high-affinity choline transporter, and provide an experimental platform for further studies of this permease family.

Prokaryotic Solute/Sodium Symporters: Versatile Functions and Mechanisms of a Transporter Family.

The solute/sodium symporter family (SSS family; TC 2.A.21; SLC5) consists of integral membrane proteins that use an existing sodium gradient to drive the uphill transport of various solutes, such as sugars, amino acids, vitamins, or ions across the membrane. This large family has representatives in all three kingdoms of life. The human sodium/iodide symporter (NIS) and the sodium/glucose transporter (SGLT1) are involved in diseases such as iodide transport defect or glucose-galactose malabsorption. Moreover, the bacterial sodium/proline symporter PutP and the sodium/sialic acid symporter SiaT play important roles in bacteria-host interactions. This review focuses on the physiological significance and structural and functional features of prokaryotic members of the SSS family. Special emphasis will be given to the roles and properties of proteins containing an SSS family domain fused to domains typically found in bacterial sensor kinases.

Interfacility transports by emergency medical services in the United States: Estimates from the National Hospital Ambulatory Medical Care Survey.

OBJECTIVE: To describe characteristics of encounters in U.S. emergency departments (EDs) brought by interfacility transport by emergency medical services (EMS) from other EDs or urgent care settings. METHODS: We performed a cross-sectional study of the National Hospital Ambulatory Medical Care Survey, a multistage probability survey of nonfederal of visits to U.S. EDS. We evaluated patients who were brought to the ED as an interfacility transport by EMS from another ED or urgent care setting between 2014 and 2017. We report demographics, clinical characteristics and treatment factors of ED encounters brought interfacility transport and assessed factors associated with discharge from the receiving ED. RESULTS: Of 562.9 million ED encounters during the assessed period, 4.5 million were brought by interfacility transport by EMS (1.1 million per year). This represented 0.8% (95% CI 0.6-1.0%) of all ED encounters and 5.3% (95% CI 4.4-6.3%) of ED encounters transported by EMS. Most encounters brought by interfacility transport were adults (85%) who were publicly insured (62%). 39% had at least one abnormal vital sign. Most encounters received diagnostic testing (84%) and were seen within 30 min of presentation (61%). 54% were admitted, and 36% were discharged from the ED. Encounters without chronic complex conditions and with normal triage vital signs were associated with ED discharge (p < 0.01). DISCUSSION: Interfacility transports between EDs transported by EMS account for <1% of ED encounters in the U.S. Nearly 40% of such encounters are ultimately discharged. Further research is needed to identify a low-risk cohort among patients in need of secondary transport.

Retrospective Evaluation of Risk Factors for Pediatric Secondary Transport.

OBJECTIVE: Emergency medical services (EMS) typically transports patients to the nearest emergency department (ED). After initial presentation, children who require specialized care must undergo secondary transport, exposing them to additional risks and delaying definitive treatment. EMS direct transport protocols exist for major trauma and certain adult medical conditions, however the same cannot be said for pediatric medical conditions or injuries that do not meet trauma center criteria ('minor trauma'). To explore the utility of such future protocols, we sought to first describe the pediatric secondary transport population and examine prehospital risk factors for secondary transport. METHODS: Pediatric secondary transport patients aged 0-18 years were identified. Patients meeting state EMS trauma protocol criteria or who were clinically unstable were excluded. Data were abstracted by chart review of EMS, community hospital ED, and specialty hospital records. Patients were compared to control patients with similar conditions who did not require secondary transport. RESULTS: This study identified 211 medical or minor trauma pediatric secondary transport patients between 2013 and 2014. The three most prevalent conditions were seizure (n = 52), isolated orthopedic injury (n = 49), and asthma/respiratory distress (n = 27). Increased odds of secondary transport for seizure patients were associated with administration of supplemental oxygen, glucose measurement, and online medical direction; for isolated orthopedic injuries, online medical direction; and for asthma/respiratory distress, administration of supplemental oxygen, and online medical direction. Decreased odds of secondary transport for seizure patients were associated with a higher GCS; for isolated orthopedic injuries, increased age and oxygen saturation; and for asthma/respiratory distress, administration of albuterol only. CONCLUSIONS: Children with seizures, isolated orthopedic injuries, and asthma/respiratory distress comprised the majority of the medical or minor trauma pediatric secondary transport population. Each of those conditions had specific risk factors for secondary transport. This study's results provide information to guide future prospective studies and the development of direct transport protocols for those populations.

Glu-311 in External Loop 4 of the Sodium/Proline Transporter PutP Is Crucial for External Gate Closure.

The available structural information on LeuT and structurally related transporters suggests that external loop 4 (eL4) and the outer end of transmembrane domain (TM) 10' participate in the reversible occlusion of the outer pathway to the solute binding sites. Here, the functional significance of eL4 and the outer region of TM10' are explored using the sodium/proline symporter PutP as a model. Glu-311 at the tip of eL4, and various amino acids around the outer end of TM10' are identified as particularly crucial for function. Substitutions at these sites inhibit the transport cycle, and affect in part ligand binding. In addition, changes at selected sites induce a global structural alteration in the direction of an outward-open conformation. It is suggested that interactions between the tip of eL4 and the peptide backbone at the end of TM10' participate in coordinating conformational alterations underlying the alternating access mechanism of transport. Together with the structural information on LeuT-like transporters, the results further specify the idea that common design and functional principles are maintained across different transport families.

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh.

GltPh from Pyrococcus horikoshii is a homotrimeric Na(+)-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in GltPh consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na(+) and aspartate binding to GltPh. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na(+) binds with low affinity to the apoprotein (Kd 120 mm), with a particularly low kon value (5.1 m(-1)s(-1)). At least two sodium ions bind before aspartate. The binding of Na(+) requires a very high activation energy (Ea 106.8 kJ mol(-1)) and consequently has a large Q10 value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na(+) concentration present. Binding of aspartate was not observed in the absence of Na(+), whereas in the presence of high Na(+) concentrations (above the Kd for Na(+)) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (kon of 1.4 × 10(5) m(-1)s(-1)), with low Ea and Q10 values (42.6 kJ mol(-1) and 1.8, respectively). We conclude that Na(+) binding is most likely the rate-limiting step for substrate binding.

HutT functions as the major L-histidine transporter in Pseudomonas putida KT2440.

Histidine is an important carbon and nitrogen source of γ-proteobacteria and can affect bacteria-host interactions. The mechanisms of histidine uptake are only partly understood. Here, we analyze functional properties of the putative histidine transporter HutT of the soil bacterium Pseudomonas putida. The hutT gene is part of the histidine utilization operon, and the gene product belongs to the amino acid-polyamine-organocation (APC) family of secondary transporters. Deletion of hutT severely impairs growth of P. putida on histidine, suggesting that the encoded transporter is the major histidine uptake system of P. putida. Transport experiments with cells and purified and reconstituted protein indicate that HutT functions as a high-affinity histidine : proton symporter with high specificity for the amino acid. Substitution analyses identified amino acids crucial for HutT function.

Hydrogen-Deuterium Exchange Mass-Spectrometry of Secondary Active Transporters: From Structural Dynamics to Molecular Mechanisms.

Membrane transporters allow the selective transport of otherwise poorly permeable solutes across the cell membrane and thus, play a key role in maintaining cellular homeostasis in all kingdoms of life. Importantly, these proteins also serve as important drug targets. Over the last decades, major progress in structural biology methods has elucidated important structure-function relationships in membrane transporters. However, structures obtained using methods such as X-ray crystallography and high-resolution cryogenic electron microscopy merely provide static snapshots of an intrinsically dynamic, multi-step transport process. Therefore, there is a growing need for developing new experimental approaches capable of exploiting the data obtained from the high-resolution snapshots in order to investigate the dynamic features of membrane proteins. Here, we present the basic principles of hydrogen-deuterium exchange mass-spectrometry (HDX-MS) and recent advancements in its use to study membrane transporters. In HDX-MS experiments, minute amounts of a protein sample can be used to investigate its structural dynamics under native conditions, without the need for chemical labelling and with practically no limit on the protein size. Thus, HDX-MS is instrumental for resolving the structure-dynamic landscapes of membrane proteins in their apo (ligand-free) and ligand-bound forms, shedding light on the molecular mechanism underlying the transport process and drug binding.

A New Critical Conformational Determinant of Multidrug Efflux by an MFS Transporter.

Secondary multidrug (Mdr) transporters utilize ion concentration gradients to actively remove antibiotics and other toxic compounds from cells. The model Mdr transporter MdfA from Escherichia coli exchanges dissimilar drugs for protons. The transporter should open at the cytoplasmic side to enable access of drugs into the Mdr recognition pocket. Here we show that the cytoplasmic rim around the Mdr recognition pocket represents a previously overlooked important regulatory determinant in MdfA. We demonstrate that increasing the positive charge of the electrically asymmetric rim dramatically inhibits MdfA activity and sometimes even leads to influx of planar, positively charged compounds, resulting in drug sensitivity. Our results suggest that unlike the mutants with the electrically modified rim, the membrane-embedded wild-type MdfA exhibits a significant probability of an inward-closed conformation, which is further increased by drug binding. Since MdfA binds drugs from its inward-facing environment, these results are intriguing and raise the possibility that the transporter has a sensitive, drug-induced conformational switch, which favors an inward-closed state.

The fascinating but mysterious mechanistic aspects of multidrug transport by MdfA from Escherichia coli.

MdfA is an interesting member of a large group of secondary multidrug (Mdr) transporters. Through genetic, biochemical and biophysical studies of MdfA, many challenging aspects of the multidrug transport phenomenon have been addressed. This includes its ability to interact with chemically unrelated drugs and how it utilizes energy to drive efflux of compounds that are not only structurally, but also electrically, different. Admittedly, however, despite all efforts and a recent pioneering structural contribution, several important mechanistic issues of the promiscuous capabilities of MdfA still seek better molecular and dynamic understanding.

Structure of the SLC4 transporter Bor1p in an inward-facing conformation.

Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate uptake in plants. The SLC4 family is more distantly related to members of the Amino acid-Polyamine-organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg. Their mechanism generally involves relative movements of two domains: a core domain that binds substrate and a gate domain that in many cases mediates dimerization. To shed light on conformational changes governing transport by the SLC4 family, we grew helical membrane crystals of Bor1p from Saccharomyces mikatae and determined a structure at ∼6 Šresolution using cryo-electron microscopy. To evaluate the conformation of Bor1p in these crystals, a homology model was built based on the related anion exchanger from red blood cells (AE1). This homology model was fitted to the cryo-EM density map using the Molecular Dynamics (MD) Flexible Fitting method and then relaxed by all-atom MD simulation in explicit solvent and membrane. Mapping of water accessibility indicates that the resulting structure represents an inward-facing conformation. Comparisons of the resulting Bor1p model with the X-ray structure of AE1 in an outward-facing conformation, together with MD simulations of inward-facing and outward-facing Bor1p models, suggest rigid body movements of the core domain relative to the gate domain. These movements are consistent with the rocking-bundle transport mechanism described for other members of the APC superfamily.

Mechanism of Na(+)-dependent citrate transport from the structure of an asymmetrical CitS dimer.

The common human pathogen Salmonella enterica takes up citrate as a nutrient via the sodium symporter SeCitS. Uniquely, our 2.5 Å x-ray structure of the SeCitS dimer shows three different conformations of the active protomer. One protomer is in the outside-facing state. Two are in different inside-facing states. All three states resolve the substrates in their respective binding environments. Together with comprehensive functional studies on reconstituted proteoliposomes, the structures explain the transport mechanism in detail. Our results indicate a six-step process, with a rigid-body 31° rotation of a helix bundle that translocates the bound substrates by 16 Å across the membrane. Similar transport mechanisms may apply to a wide variety of related and unrelated secondary transporters, including important drug targets.

Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms.

Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to one or other side of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (a)symmetry of these systems has been successfully used as a bioinformatic tool, called "repeat-swap modeling" to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that nucleoside transport also occurs via an elevator-like mechanism.

Divide and conquer: processive transport enables multidrug transporters to tackle challenging drugs.

Multidrug transporters are membrane proteins that catalyze efflux of antibiotics and other toxic compounds from cells, thereby conferring drug resistance on various organisms. Unlike most solute transporters that transport a single type of compound or similar analogues, multidrug transporters are extremely promiscuous. They transport a broad spectrum of dissimilar drugs and represent a serious obstacle to antimicrobial or anticancer chemotherapy. Many challenging aspects of multidrug transporters, which are unique, have been studied in detail, including their ability to interact with chemically unrelated drugs, and how they utilize energy to drive efflux of compounds that are not only structurally but electrically different. A new and surprising dimension of the promiscuous nature of multidrug transporters has been described recently: they can move long molecules through the membrane in a processive manner.

O transporte inter-hospitalar do doente crítico: documentação e continuidade de cuidados / The inter-hospital transport of the critically ill patient: documentation and continuity of care

O transporte inter-hospitalar do doente crítico é um procedimento frequente face à necessidade de meios complementares de diagnóstico e terapêutica com elevado nível de diferenciação. Embora represente um risco adicional para o doente, o planeamento adequado é fundamental para a diminuição da ocorrência de complicações. Partindo de uma experiência profissional enquanto enfermeira envolvida no processo de transporte inter-hospitalar do doente crítico e de uma avaliação e reflexão enquanto estudante de mestrado em estágio num serviço de urgência, decidiuse por estudar a documentação dos cuidados de enfermagem durante o transporte inter-hospitalar do doente crítico, como garantia da continuidade dos cuidados. Optou-se pela metodologia de projeto, partindo de um diagnóstico de situação realizado através de um questionário para identificação de incidentes/ocorrências clínicos e técnicos mais frequentes durante o processo de transporte. Participaram no projeto de intervenção em serviço 64 enfermeiros do Serviço de Urgência e da Unidade de Cuidados Intermédios Polivalente de um Hospital da Região Norte (N=64). Os dados obtidos foram sujeitos a análise descritiva, sendo posteriormente apresentados aos enfermeiros-chefes e à equipa de enfermagem. De forma genérica, pode dizer-se que os incidentes/ocorrências clínicos mais reportados foram o aumento/diminuição da tensão arterial, a alteração do traçado eletrocardiográfico e a diminuição da saturação periférica de oxigénio, destacando-se a trepidação, a limitação do espaço para aceder ao doente e aos equipamentos e o desconhecimento dos materiais e dos equipamentos de transporte como os incidentes/ocorrências técnicos mais frequentes. Com base nestes dados, foi elaborado um documento que permite ao enfermeiro, envolvido no transporte do doente crítico, documentar o processo de tomada de decisão e garantir a continuidade de cuidados. Salienta-se, ainda, que a criação de uma equipa de transporte especializada, meios de transporte mais adequados, bem como a necessidade de documentação e registos, de melhoria no planeamento e formação na área, foram sugestões apresentadas pelos enfermeiros como importantes e necessárias para a melhoria do transporte inter-hospitalar do doente crítico. A possibilidade de registo das intervenções de enfermagem, aliadas às sugestões que os enfermeiros apresentaram, podem ser um contributo para a melhoria dos cuidados de enfermagem prestados ao doente crítico durante o transporte inter-hospitalar.

The interhospital transportation of the critical patient is a frequent procedure due to the need for complementary diagnostic and therapeutic means with a high level of differentiation. Although it poses an additional risk to the patient, adequate planning is critical to reducing the occurrence of complications. Starting from a professional experience as a nurse involved in the interhospital transportation process of the critically ill patient and an evaluation and reflection as a master's student in an emergency department, it was decided to study the documentation of nursing care during interhospital transportation of the critical patient, as a guarantee of continuity of care. We chose the project methodology, based on a situation diagnosis performed through a questionnaire to identify the most frequent clinical and technical incidents/occurrences during the transportation process. Sixty-four nurses from the Emergency Department and the Polyvalent Intermediate Care Unit of a Hospital in the Northern Region (N=64) participated in the project. The data were subjected to descriptive analysis and later presented to the head nurses and the nursing team. Generally, the most reported clinical incidents/occurrences were increased/decreased blood pressure, altered electrocardiographic tracing and decreased peripheral oxygen saturation, especially the vibration, limiting the access space and equipment and the lack of knowledge of materials and transportation equipment such as the most frequent technical incidents/occurrences. Based on these data, a document was developed that allows the nurse, involved in the transportation of the critical patient, to document the decision-making process and ensure continuity of care. It should also be noted that the creation of a specialized transport team, more adequate means of transport, and the need for documentation and records to improve planning and training in the area were suggestions presented by nurses as important and necessary for the improvement interhospital transport of the critical patient. The possibility of registration of nursing interventions combined with the suggestions that the nurses presented may be a contribution to the improvement of the nursing care provided to the critical patient in the interhospital transport.